Spermatogenic cells are characterized by the presence of long-chain polyunsaturated fatty acids in their glycerolipids and hormone-coordinated cellular proliferation. The first and committed step in glycerolipid synthesis is the acylation of glycerol-3-phosphate, catalyzed by the enzyme glycerol-3-phosphate acyltransferase (GPAT). In mammalian cells, four GPAT isoforms have been cloned and characterized, each one with different kinetic properties, tissue and cell locations. The long-term goal of both groups collaborating in this project is to determine the cellular and metabolic functions of each GPAT isoform. Mitochondrial GPAT2 is expressed primarily in testis and secondarily in brain and some cancer cells. GPAT2-expressing tissues, like brain and cancer cells share some of the characteristics of spermatogenic cells, including an increased content of polyunsaturated fatty acids and rapid proliferation, respectively. The hypothesis underlying this project is that GPAT2 contributes to glycerophospholipid synthesis in tissues like testis and brain that have a high content of PUFA and that it catalyzes the synthesis of a signaling lipid molecule that helps to coordinate cellular proliferation. The specific aims of this project are: Aim 1: Determine whether the function and regulation of GPAT2 is related to the preferential esterification of polyunsaturated fatty acids in glycerolipids. Aim 2: Determine the function of GPAT2 in testis and cancer cells related to cellular proliferation and its mechanism. To test these hypotheses, recombinant mouse GPAT2 will be expressed in cultured mammalian cells, and glycerolipid synthesis will be assessed with different fatty acid substrates. Transcriptional regulation of GPAT2 will be determined in cells exposed to spermatogenesis-associated hormones, and by expressing the GPAT2 promoter region in a reporter-gene construct. The nuclear receptors involved in GPAT2 regulation will be determined by super-shift-gel electrophoresis assays. Experiments proposed to study GPAT2 activity related to cellular proliferation includes the mass-spectrometry quantification of lysophosphatidic acid, synthesized de novo as a signaling molecule, and the detection of activated intermediates of the mTOR signaling pathway in GPAT2 overexpressing cells. The physiological function of GPAT2 activity will be studied by post- transcriptionally silencing the enzyme's expression both in vivo and in vitro. Using GPAT2-targeted small- interfering RNA in sexually developing mice, sperm production and motility will be determined. The effect of GPAT2 silencing on the tumor-producing capability of cancer cells will also be determined. These studies will improve our understanding of the molecular mechanisms that govern sperm maturation and cellular proliferation, which are important to both reproduction and the proliferation of cancer cells. This research will be done in collaboration with NIH-funded investigator Dr. Rosalind A. Coleman, Department of Nutrition, University of North Carolina at Chapel Hill, USA. PUBLIC HEALTH RELEVANCE: This project is related to two major global health problems: human reproduction and cancer. Increasing our knowledge about the enzymes and molecular mechanisms involved in normal sperm maturation and in cancer growth and proliferation may improve the design of new inhibiting drugs to treat cancer or be used as a contraceptive method, or as activating drugs to treat infertility.